In general, gene medicines are negatively charged due to their structural characteristic, and thus many studies have been conducted on in vivo delivery system, which is a complex prepared by associating negative charge of small interfering RNA or plasmid DNA with positive charge of cationic polymers such as polyethyleneimine and polylysine, or cationic phospholipid nanoparticles.
Cationic phospholipid nanoparticles are disclosed in U.S. Pat. No. 5,858,784 and US Patent Publication NO. 20060008910A1, in which cationic lipids are mixed with phospholipids in a predetermined ratio to prepare particles such as cationic liposome, the particles are mixed with nucleic acid to prepare a complex of cationic phospholipids particles and nucleic acid, and then the complex is introduced into a cell line to improve gene expression. Cationic polymers have been also studied as a gene delivery vehicle, which are disclosed as follows: DEAE dextran, polylysine having repeating lysine units, and polyethyleneimine having repeating ethyleneimine units in U.S. Pat. No. 6,020,457, poly-amino-ester in US Patent Publication NO. 20040071654A1, and a biodegradable cationic copolymer in US patent Publication NO. 20060093674A1.
As a gene delivery vehicle, the synthetic polymers are advantageous in that they are easily prepared, not limited by the size of gene to be introduced, generate fewer side effects that may be induced by immunogenic viral surface protein upon repeated administration, cause no safety problems due to viral genes, and require lower production cost in a commercial process, as compared to viral vectors including lentiviral, adenoviral and retroviral vectors. However, there are drawbacks in that the delivery systems using such cationic polymers have lower transfection efficiency as compared to viral vectors that are effectively transferred via cell surface receptors, and induce cytoxicity (J. Control. Release (2006) 114, 100-109). Another drawback of the cationic polymer mediated gene transfer is that it does not greatly prolong the half life in blood (Gene Ther. (2001) 8, 1857-1592).
Among cationic polymers, chitosan has been studied as a promising candidate for gene delivery because it has several advantages such as industrial availability and biocompatibility. However, it has to overcome such problems as solubility and low transfection efficiency. In order to improve such problems, U.S. Pat. No. 6,730,742 (2004) discloses that chitosan is conjugated to polyethylene glycol to prepare a polymer conjugate, and the polymer conjugate is used as a gene delivery system for mucosal administration. However, there is a limitation in that only the addition of polyethylene glycol does not significantly improve the gene transfer efficiency.
Accordingly, the present inventors have made an effort to improve gene transfer efficiency and stability in blood by using chitosan. They found that a double conjugate that is prepared by linking a cationic polyamine to chitosan or a triple conjugate that is prepared by additionally linking a biocompatible polyethylene glycol (PEG) to the double conjugate is used to reduce cytotoxicity and improve gene transfer efficiency or retention time in blood, thereby completing the present invention.